Combinatorial Assembly and Optimisation of Designer Cellulosomes - A Galactomannan Case Study

Background: Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome. Results: VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers. Conclusion: We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS-Hook-FHIP complexes

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ~40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS-Hook-FHIP ('FHF') cargo adaptor complex links dynein to cargo in mammals and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein 'interactome' of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 co-localize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS-Hook-FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS-Hook-FHIP complexes

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ~40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS-Hook-FHIP (FHF) cargo adaptor complex links dynein to cargo in mammals and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein interactome of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 co-localize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS-Hook-FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO2 capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO2, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO2, avoiding fossil carbon or competition with food production.

Chromatin accessibility combined with enhancer clusters activation mediates heterogeneous response to dexamethasone in myeloma cells

Glucocorticoids (GC) effects occur through binding to the GC receptor (GR) which, once translocated to the nucleus, binds to GC response elements (GREs) to activate or repress target genes. Among GCs, dexamethasone (Dex) is widely used in treatment of multiple myeloma (MM), mainly in combination regimens. However, despite a definite benefit, all patients relapse. Moreover, while GC efficacy can be largely attributed to lymphocyte-specific apoptosis, its molecular basis remains elusive. To determine the functional role of GR binding in myeloma cells, we generated bulk and single cell multi-omic data and high-resolution contact maps of active enhancers and target genes. We show that a minority (6%) of GR binding sites are associated with enhancer activity gains and increased interaction loops. We find that enhancers contribute to regulate gene activity through combinatorial assembly of large stretches of enhancers and/or enhancer cliques. Furthermore, one enhancer, proximal to GR-responsive genes, is predominantly associated with increased chromatin accessibility and higher H3K27ac occupancy. Finally, we show that Dex exposure leads to co-accessibility changes between predominant enhancer and other regulatory regions of the interaction network. Notably, these epigenomic changes are associated with cell-to-cell transcriptional heterogeneity. As consequences, BIM critical for GR-induced apoptosis and CXCR4 protective from chemotherapy-induced apoptosis are rather upregulated in different cells. In summary, our work provides new insights into the molecular mechanisms involved in Dex escape.

DNA-based data storage via combinatorial assembly

Persistent data storage is the basis of all modern information systems. The long-term value and volume of data are growing at an accelerating rate and pushing extant storage systems to their limits. DNA offers exciting potential as a storage medium, but no practical scheme has been proposed to date that can scale beyond narrow-band write rates. Here, we demonstrate a combinatorial DNA data encoding scheme capable of megabits per second write speeds. The system relies on rapid, combinatorial assembly of multiple smaller DNA parts that are dispensed through inkjet printing. To demonstrate this approach, we wrote approximately 25 kB of information into DNA using our system and read the information back out with commercially available nanopore sequencing. Moreover, we demonstrate the ability to replicate and selectively access the information while it is in DNA, opening up the possibility of more sophisticated DNA computation.

Survival associated with mutations in SWI/SNF chromatin remodeling complex genes.

3643 Background: The SWI/SNF (SWitch/Sucrose NonFermentable) chromatin remodeling complex (CRC) - a combinatorial assembly of products from multiple genes - alters histone/DNA interactions and thereby impacts transcription, DNA replication/repair, and cell division. Studies suggest that over 20% of human cancers contain mutations in at least one SWI/SNF gene, implying that it is the most highly mutated CRC in human cancer. To address existing knowledge gaps, we sought to evaluate the association between SWI/SNF mutations and overall survival (OS). Methods: We identified adult cancer patients who consented to have OncoPanel testing (Dana-Farber/Brigham & Women’s Hospital’s next generation sequencing platform) from June 2013-August 2019. These data were merged with institutional electronic health records and National Death Index vital status. We determined mutation frequency and co-occurrence for the nine SWI/SNF genes included in OncoPanel (ARID1A, ARID1B, ARID2, BCL11B, PBRM1, SMARCA4, SMARCB1, SMARCE1, and SS18). We assessed the association between mutation and OS (from time of OncoPanel testing) for cancers with at least 500 analyzed and 20 mutated cases, controlling for age and TP53 status. Exploratory analyses were conducted using cBioPortal and SAS (no multiple comparison adjustment). Results: Among 25,434 samples from 24,648 patients, a mutation in at least one evaluated SWI/SNF gene was identified in 26% of cases (ARID1A 10.5%, ARID1B 7.2%, SMARCA4 5.5%, PBRM1 4.9%, ARID2 4.8%, BCL11B 3.5%, SMARCE1 1.1%, SMARCB1 1.0%, and SS18 0.7%). The most frequently mutated cancers included small bowel (52%), endometrial (49%), ampullary (48%) and bladder (45%). Co-occurrence was common (30 of 36 potential gene-pairs), with the largest associations (odds ratio; all P < .05) seen for SMARCB1:BCL11B (4.19), ARID1B:BCL11B (3.87), ARID2:BCL11B (3.85), and SMARCA4:BCL11B (3.78). Associations between having a mutation and OS were seen for the following cancers/genes (odds ratio; all P < .05): ARID1A (colorectal 0.72, pancreatic 1.46), ARID1B (melanoma 0.32), SMARCA4 (esophagogastric 1.48, non-small cell lung 1.89, ovarian 0.43), SMARCB1 (non-small cell lung 2.04), and SS18 (soft tissue sarcoma 2.06). Conclusions: Mutations in SWI/SNF genes are widespread, with mutation rates varying by cancer type. Co-occurrence was common, especially with BCL11B. Associations with OS were both favorable and unfavorable, with variability seen by gene and cancer type. Future research should explore the mechanisms by which mutations in SWI/SNF genes influence treatment response/OS.

A Penicillium rubens platform strain for secondary metabolite production

We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.

High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

ABSTRACTDespite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5’ untranslated regions (5’ UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5’UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5’ UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5’ UTRs. A total of ∼12,000 5’ UTRs were then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identified three synthetic 5’ UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5’ UTRs enabled even higher protein expression than obtained with each individual 5’ UTR. In summary, we demonstrate that high-throughput screening of 5’ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.